Abstract

G-protein coupled receptors (GPCRs) are transmembrane proteins that are found at the plasma membrane in eukaryotes, where they direct responses to many environmental stimuli and control many aspects of cellular function. The similarity between pheromone signaling in yeast and GPCR-mediated signaling in humans has allowed the development of genetic and chemical high-throughput screening to isolate agonists or antagonists of GPCRs using yeast biosensors with functionally coupled heterologous GPCRs. This project aims to improve the signaling sensitivity of heterologous GPCRs in engineered yeast by introducing positive feedback loops (PBLs) consisting of primary signal-induced expression of critical regulators of the signaling pathway, such as adaptor protein Ste50 and scaffold protein Ste5. Ste50 and Ste5 were upregulated through overexpression of an inducible promoter that is controlled by the mating signal. A comparative analysis through a long-term assay and a short-term assay was used to test if the weak signaling of a GPCR can be amplified when compared to the wildtype. Our results show that for long-term assays the addition of Ste50 through the positive feedback loop supported increased reporter signaling whereas Ste5 alone and in combination with Ste50 caused Far1-independent cell cycle arrest. In short-term assays, the combined positive feedback loop system, Ste50 and Ste5 demonstrated higher levels of signaling, as compared to either one alone or the wildtype. This study presents a possible solution to increasing the reporter output of the yeast pheromone pathway with greater sensitivity through controlled positive feedback loops of two positive regulators.